Drosophila Cell Culture

I. MEDIUM

A. S2, S2C, S2*, S2R+, S3, Kc(167), DL1, & DL2 cells:

Schneider''s/ 10% FBS/ PS
450 ml Schneider''s medium (Gibco #11720-034)
(pour off 50 ml into Falcon to save as serum-free)
***[S2R+ and Kc cells will not grow in Schneider''s medium from Sigma]
50 ml Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 ml 1:100 Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
Sterile filter (0.2 µ) Store at 4°C - do not freeze

B. ML-DmBG2 & ML-DmBG6 cells:

M3/10% FBS/PS/Insulin
450 ml Shields and Sang M3 insect medium (Sigma #S8523)
(pour off 50 ml into Falcon to save as serum-free)
50 ml Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 ml 1:100 Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
20 ug/ml Insulin (Sigma #I6634)
Sterile filter (0.2 µ) Store at 4°C - do not freeze

C. Clone8 (CL8) cells:

Complete M3 Medium
Shields and Sang M3 insect medium (Sigma #S3652)
2% Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
2.5% Fly extract (see below)
0.125 IU/ml (0.5mg/ml) Insulin (Sigma #I6634)
1x Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
Sterile filter (0.2 µ)

Heat Inactivating Fetal Bovine Serum

1. Thaw the Fetal Bovine Serum on a shaker at 2-8°C (overnight)
2. Pre-heat a waterbath to a temperature of 56°C. (Make sure the water covers all of the serum in the bottle)
3. Heat the serum for 30 minutes in the 56°C bath.
4. Allow serum to cool to room temperature before adding to cells.

Insulin Stock (100x)

1. Dissolve 10mg (25IU) in 0.5ml of 0.01N HCL.
2. Heat in 37°C to dissolve for few minutes.
3. Add 19.5mL of M3 Media to bring volume up to 20mL.
4. Filter Sterilize with a 0.22µm filter.
5. Alliquot and store stock at -20°C. Working stock may be kept at 4°C for 4-5 weeks.

Fly Extract

1. Start with a collection of 30-75g of healthy OreR, CanS, or yw flies. These can be collected and frozen, dry at -80° C. Warm centrifuge to room temperature.
2. To make 200ml extract, use 30g of frozen flies.
   To make 400ml extract, use 60g of frozen flies.
   To make 500ml extract, use 75g of frozen flies.
3. Add weighed out flies to blender. Add appropriate amount of Shields and Sang M3 medium. Blend at medium to high speed until it looks as if all the flies have been lysed and the liquid is reddish from dispersed eye pigment.
4. Transfer liquid with fly carcasses to 250ml gasketted centrifuge bottles. Spin at 2600rpm in a GSA rotor for 20 minutes.
5. Aspirate the fly bodies and oily layer off the top of the bottle.
6. Transfer liquid to new 250ml gasketted centrifuge bottles. Spin again at 3000rpm in a GSA rotor for 30 minutes
7. Again aspirate off the oily layer on top. Transfer liquid to new centrifuge bottles.
8. Heat Inactivate the fly extract by placing centrifuge bottles into a 60°C water bath for 30-40 minutes. You will see a precipitate form.
9. Centrifuge the extract at 3200 rpm in a GSA rotor for 45 minutes. Precipitated material will form a grayish pellet.
10. Filter the supernate. Filter through a 0.22µm filter in a tissue culture hood. For the first filter pass, place all of the glass pre-filters provided in the filter unit box on top of the 0.22µm filter. Hold them down with 2 pipettes as you pour the supernate into the filter unit. The fly extract tends to clog the filters, so you may need to use more than one filter unit. Once all the extract has been through at least one 0.22µm filter, aliquot into 12.5mL aliquots and freeze in liquid nitrogen. Store at -20°C.

II. GROWTH CONDITIONS

Cells grow @ 23-25°C and @ RT
Do not need CO²
Split every 3-4 days to maintain
Density sensitive - die if too dense or too dilute

III. MAINTENANCE

Split one flask of cells (most recent date) into two new T-75 flasks (VWR# BD353136) when culture is confluent. Keep other flask as a backup. Monitor growth status by microscopy before splitting and decide whether to adjust recommended dilution factor accordingly.

A. Semi-adherent cell lines - will stick to new flask but come loose over time

S2*	3-4 days	1:3 - 1:4
S2c	3-4 days	1:3 - 1:4
Kc(167)	3-4 days	1:3 - 1:4
l(2)mbn	3-4 days	1:3 - 1:4

B. Adherent cell lines

DL1	3-4 days	1:8 (1:5 - 1:10)
DL2	3-4 days	1:8 (1:5 - 1:10)
S3	3-4 days	1:3
S2R+	3-4 days	1:3 - 1:4
CL8	4-5 days	1:5-1:10
BG2	4-5 days	1:3-1:4
BG6	4-5 days	1:3-1:4

SL2, S2C, S2*

Detach cells from the flask either by banging or scraping
Pipet cells up and down about 10 times to resuspend cells and separate clumps
Aliquot cells into new flasks accordingly

DL2, DL1, S2R+, Kc167, Clone 8, S3

Protocol 1:
Remove all medium
Scrape cells with scraper
Resuspend cells with 10mL of fresh medium, pipetting up and down about 10 times
Aliquot cells into new flasks accordingly

Protocol 2:
Remove medium
Wash in PBS to remove any serum
Add 5 ml trypsin; let sit 5-10''
Bang cells off flask.
Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
Spin down cells at 1200-1400 rpm for 5 min.
Resuspend cells in serum medium.
Aliquot cells into new flasks accordingly

Protocol 3:
Detach cells from the flask either by banging or scraping
Pipet cells up and down about 10 times to resuspend cells and separate clumps
Aliquot cells into new flasks accordingly

BG2

1. Remove medium (save and filter through syringe filter for use as "conditioned medium").
2. Wash in PBS to remove any serum
3. Add 5 ml trypsin; let sit 5-10''
4. Bang cells off flask.
5. Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
6. Spin down cells at 1200-1400 rpm for 5 min.
7. Resuspend cells in conditioned medium.
8. Aliquot cells into new flasks accordingly

Can also use Accutase instead of trypsin.

IV. FREEZING

1. Grow cells to subconfluencey - approximately 1-2 x 10⁷ cells/ml.
2. Label appropriate # of cryovials.
3. Remove cells from flask (trypsinize and resuspend in medium if necessary).
4. Spin cells at 1200 rpm 5''
5. Aspirate off medium.
6. Resuspend at approximately 1.1 x 10⁷ cells/ml in freezing medium:
     FBS + 10% DMSO, filtered OR Complete medium + 10% DMSO, filtered
7. Aliquot 1ml cell suspension/cryovial.
8. Put vials in foam box, tape closed, and place in -70°C.
9. After a few days, transfer vials to LN₂ for long term storage.

V. THAWING

1. Prepare 15ml conical tube with 5ml medium.
2. Thaw cells quickly in water bath.
3. Just before cells are completely thawed, decontamiate the outside of the tube with 70% EtOH.
4. Transfer the cells to the conical tube with 5ml medium.
5. Spin at 1200rpm 5''.
6. Aspirate and resuspend cells in 5ml medium.
7. Plate in a T25 flask.
8. Watch daily. Initially, cells may need to be split at irregular intervals.

VI. COUNTING

1. Remove ~0.5 ml cells from flask (trypsinize cells if necessary) into eppendorf.
2. Dilute cells into Trypan Blue according to estimated density.
3. (confluent cultures try 30:70 µl to 50:50 µl of cells:TB).
4. Transfer 10 - 12 µl cells to haemocytometer.
5. Count cells in opposite corners (~100-200/ large square).
6. Calcuation:
       (count #1 + count #2) / 2 x (dilution factor) x 10⁴ = X cells/ml (usually ~1-10 x 10⁷)